Bacteriophage T7 is a virulent bacteriophage that infects Escherichia coli. It belongs to a class of bacteriophages that specify relatively simple RNA polymerases that selectively transcribe the DNA of their own bacteriophage but do not transcribe unrelated DNAs Hausmann, Current Topics in Microbiology and Immunology, 75, 77-110 (1976); Korsten, et al., J. Gen. Virol., 43, 57-73 (1979); Towle, et al., J. Biol. Chem., 250, 1723-1733 (1975); Butler and Chamberlin, J. Biol. Chem., 257, 5772-5778 (1982); Chamberlin, et al., Nature, 228, 227-231 (1970); Dunn, et al., Nature New Biology, 230, 94-96 (1971)!. The T7 bacteriophage has been the subject of extensive scientific inquiry, in part because of its simple yet highly specific RNA polymerase. The genetic organization of T7 and the pattern of gene expression during infection are well understood, and the entire nucleotide sequence of T7 DNA is known (Studier and Dunn, Cold Spring Harbor Symp. Quant. Biol., 47, 999-1007 (1983); Dunn and Studier, J. Mol. Biol., 166, 477-535 (1983); Moffatt, et al., J. Mol. Biol., 173, 265-269 (1984)!. These papers review and provide further references to a considerable body of work on T7 RNA polymerase.
T7 RNA polymerase, the product of T7 gene 1, is a protein produced early in T7 infection; it is a single-chain enzyme with a molecular weight close to 100,000. It appears that the basis for the selectivity of the T7 RNA polymerase is the interaction of the RNA polymerase with a relatively large promoter sequence, a sequence large enough that it is unlikely to be found by chance in any unrelated DNA. In the case of T7, the highly conserved promoter sequence appears to consist of approximately 23 continuous base pairs, which includes the start site for the RNA chain. If exact specification of even as few as 15 of these base pairs were required for initiation of chains, chance occurrence of a functional promoter would be expected less than once in a billion nucleotides of DNA.
The stringent specificity of T7-like RNA polymerases for their own promoter sequences is used by these phages to direct all transcription and replication to their own DNAs during infection. After the phage RNA polymerase is made, other phage gene products inactivate the host RNA polymerase, leaving all transcription in the cell directed by the phage enzyme.
T7 RNA polymerase is very efficient at transcribing DNA from its own promoters, and elongates RNA chains about five times faster than does E. coli RNA polymerase Golomb and Chamberlin, J. Biol. Chem., 249, 2858-2863 (1974)!. Termination signals for T7 RNA polymerase do not seem to occur very frequently, and termination is usually not very efficient McAllister, et al., J. Mol. Biol., 153, 527-544 (1981)!.
Their selectivity, activity, and ability to produce complete transcripts make T7 RNA polymerase and the equivalent RNA polymerases from T7-like phages useful for a variety of purposes. T7 RNA polymerase and SP6 RNA polymerase have been purified from infected cells and have been used to produce RNAs for translation in vitro Dunn and Studier, J. Mol. Biol., 166, 477-535 (1983)!, substrates for splicing Green, et al., Cell, 32, 681-694 (1983)!, and hybridization probes Zinn, et al., Cell, 34, 865-879 (1983)!. T7 RNA polymerase made during T7 infection directs the expression of genes under control of T7 promoters in plasmids Campbell, et al., PNAS USA, 75:2276-2280 (1978); Studier and Rosenberg, J. Mol. Biol, 153:503-525 (1981); and McAllister, et al., J. Mol. Biol., 153:527-544 (1981)!, but these gene products do not accumulate to high levels because of competition from promoters in T7 DNA and because the T7 infection quickly kills the cell. It was anticipated McAllister, et al., J. Mol. Biol., 153:527-544 (1981)!, and the present invention demonstrates, that T7 RNA polymerase would also be useful for directing high-level expression of selected genes in cells. The problem in designing a high-level expression system is how to deliver active T7 RNA polymerase to a cell that contains a T7 promoter.
In the past, phage RNA polymerases like T7 RNA polymerase could be obtained only by infection with the phage from which they derive. The yield of purified RNA polymerase from infected cells is not particularly good, because the enzyme is synthesized for only a few minutes during the infection and does not accumulate to high levels. Nor is infection by these phages an efficient way to direct the transcription of non-phage genes inside the cell, because there is competition from promoters in the phage DNA itself and because the cells lyse within a short time.
Production of active T7 RNA polymerase from the cloned gene is an obvious way to obtain large amounts of enzyme for purification, and to have a source of enzyme that could be introduced into a variety of cells without the disadvantages associated with infection by T7 itself. Presumably recognizing this, other workers attempted to clone the active gene from T7 DNA but were not successful. In one report, Stahl and Zinn J. Mol. Biol., 148:481-485 (1981)! obtained a clone of the entire gene except for the last nucleotide of the termination codon. However, loss of the termination codon causes additional amino acids to be added to the carboxy terminus, and the protein produced from the clone was inactive.
The present invention discloses a successful process for cloning and expressing the T7 RNA polymerase gene, a process that can also be applied to clone the RNA polymerase genes from other T7-like phages. The same method has subsequently been applied by Tabor and Richardson, PNAS USA, 82:1074-1078 (1985) to obtain a different clone of the T7 RNA polymerase gene, and by Morris, et al., Gene, 41:193-200 (1986) to obtain a clone of the T3 RNA polymerase gene. Having a clone of the active gene enables the use of it for making large amounts of RNA polymerase for purification, and also enables the use of it to direct sustained, high-level expression of selected genes in the cells. The present invention discloses successful methods for implementing these uses of the cloned gene for T7 RNA polymerase in E. coli.